Abstract
The CRISPR RNA-guided Cas9 nuclease gene-targeting system has been extensively used to edit the genome of several organisms. However, most mutations reported to date have been are indels, resulting in multiple mutations and numerous alleles in targeted genes. In the present study, a large deletion of 105 kb in the TYR (tyrosinase) gene was generated in rabbit via a dual sgRNA-directed CRISPR/Cas9 system. The typical symptoms of albinism accompanied significantly decreased expression of TYR in the TYR knockout rabbits. Furthermore, the same genotype and albinism phenotype were found in the F1 generation, suggesting that large-fragment deletions can be efficiently transmitted to the germline and stably inherited in offspring. Taken together, our data demonstrate that mono and biallelic large deletions can be achieved using the dual sgRNA-directed CRISPR/Cas9 system. This system produces no mosaic mutations or off-target effects, making it an efficient tool for large-fragment deletions in rabbit and other organisms.
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Acknowledgments
We thank Peiran Hu at the Embryo Engineering Center for the critical technical assistance. This work was financially supported by the National Natural Science Foundation of China (Grant No. 31201080 and 31272394).
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Y. Song and L. Yuan contributed equally to this work.
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18_2016_2143_MOESM1_ESM.jpg
Figure S1. Off-target analysis of the 4 sgRNAs in KO founders. PCR and T7EI assays of the PCR products of candidate off-target sites for 4 sgRNAs in founder #301 (primer sequences listed in Table S3). No fragment was found in T7EI assays (JPEG 1693 kb)
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Figure S2. Schematic diagram of the SNP located in exon3 of TYR gene. Schematic diagram of exon3 of TYR in TYR KO rabbits and WT rabbits. The CDS region is indicated by blue rectangles; the SNP is located in a yellow circle and indicated by the arrows. WT, wild-type control; NW, New Zealand white rabbit; #301 and #106 are TYR KO rabbits (JPEG 1932 kb)
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Figure S3. Sequence diagram of POTS in TYR KO founders. Sequence diagram of 20 potential off-target sites for sgRNA1, sgRNA2, sgRNA3, and sgRNA4 showing no double curve in any sequencing diagrams. Blue area represents sequencing of the POTS (JPEG 5527 kb)
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Song, Y., Yuan, L., Wang, Y. et al. Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system. Cell. Mol. Life Sci. 73, 2959–2968 (2016). https://doi.org/10.1007/s00018-016-2143-z
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DOI: https://doi.org/10.1007/s00018-016-2143-z